JP2832760B2 - Sterilization method of immobilized enzyme - Google Patents
Sterilization method of immobilized enzymeInfo
- Publication number
- JP2832760B2 JP2832760B2 JP3075839A JP7583991A JP2832760B2 JP 2832760 B2 JP2832760 B2 JP 2832760B2 JP 3075839 A JP3075839 A JP 3075839A JP 7583991 A JP7583991 A JP 7583991A JP 2832760 B2 JP2832760 B2 JP 2832760B2
- Authority
- JP
- Japan
- Prior art keywords
- immobilized
- immobilized enzyme
- treatment
- enzyme
- ultrasonic treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Apparatus For Disinfection Or Sterilisation (AREA)
Description
【0001】[0001]
【産業上の利用分野】本発明は、超音波処理と紫外線照
射処理を併用する固定化酵素の殺菌方法に関する。本発
明の殺菌方法を用いることによって、酵素活性を低下さ
せずに固定化酵素に付着している微生物を殺菌すること
ができる。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for sterilizing an immobilized enzyme by using both an ultrasonic treatment and an ultraviolet irradiation treatment. By using the sterilization method of the present invention, microorganisms adhering to the immobilized enzyme can be sterilized without reducing the enzyme activity.
【0002】[0002]
【従来の技術】従来、物質の生産あるいは改良に広く固
定化酵素が利用されている。しかし、固定化酵素を長期
間連続して使用した場合、固定化酵素に付着した細菌な
どの微生物が増殖し、その結果、生成物の微生物汚染や
固定化酵素の活性低下などの問題が生じてくる。2. Description of the Related Art Conventionally, immobilized enzymes have been widely used for producing or improving substances. However, if the immobilized enzyme is used continuously for a long period of time, microorganisms such as bacteria attached to the immobilized enzyme will proliferate, and as a result, problems such as microbial contamination of the product and reduced activity of the immobilized enzyme will occur. come.
【0003】このような問題点を改善するため、固定化
酵素を洗浄する際に殺菌剤あるいは洗浄剤を用いること
が提案されている。例えば、固定化酵素をジオクチルジ
エチレントリアミンなどの置換ジエチレントリアミンの
希釈水溶液と接触させて殺菌する方法(特開昭56−9
2791)、微生物汚染した固定化酵素を多価アルコー
ルに浸漬して殺菌する方法(特開昭59−17587
9)、固定化酵素をラウリルベンゼンスルホン酸塩とエ
タノールを含む混合液で処理して洗浄殺菌する方法(特
開昭59−98689)などである。しかし、これらの
固定化酵素を殺菌剤や洗浄剤で洗浄殺菌する方法は、そ
の洗浄殺菌に長時間を要したり、あるいは洗浄殺菌効果
が必ずしも十分でなく、また、固定化酵素の活性を低下
させたり、洗浄殺菌後、固定化酵素に残存付着している
洗浄剤や殺菌剤を完全に除去する操作を必要とするな
ど、種々の解決をしなければならない問題があった。In order to solve such problems, it has been proposed to use a bactericide or a detergent when washing the immobilized enzyme. For example, a method in which an immobilized enzyme is sterilized by contact with a dilute aqueous solution of a substituted diethylenetriamine such as dioctyldiethylenetriamine (Japanese Patent Laid-Open No. 56-9 / 1981)
2791), a method of immersing the immobilized enzyme contaminated with microorganisms in polyhydric alcohol for sterilization (JP-A-59-17587).
9), a method in which the immobilized enzyme is treated with a mixed solution containing laurylbenzenesulfonate and ethanol and washed and sterilized (JP-A-59-98689). However, the method of washing and disinfecting these immobilized enzymes with disinfectants and detergents requires a long time for washing and disinfection, or the washing and disinfecting effect is not always sufficient, and the activity of the immobilized enzymes is reduced. There is a problem that various solutions have to be taken, such as an operation of completely removing the detergent and the bactericide remaining on the immobilized enzyme after washing and sterilization.
【0004】また、固定化酵素に紫外線を照射強度0.
5mW/cm2 以下で照射することによって、固定化酵
素の活性を低下させずに担体に付着している微生物を死
滅させる固定化酵素の殺菌方法(特開平1−22845
4)が開示されている。この方法において望ましい殺菌
効果をえるためには、固定化酵素の全面に紫外線を安定
的かつ均等に照射する必要があり、特定の殺菌装置を必
要とした。本出願人は、このような点に注目し、洗浄殺
菌反応槽内で固定化酵素を回転及び/または昇降動作を
繰り返させ、その上から紫外線を照射する固定化酵素殺
菌装置(特願平2−43893)を発明した。[0004] Further, the intensity of irradiation of the immobilized enzyme with ultraviolet light is set to 0.1.
A method for sterilizing an immobilized enzyme, which irradiates at 5 mW / cm 2 or less to kill microorganisms adhering to a carrier without lowering the activity of the immobilized enzyme (Japanese Unexamined Patent Publication No. 1-28455)
4) is disclosed. In order to obtain a desired sterilizing effect in this method, it is necessary to irradiate the entire surface of the immobilized enzyme with ultraviolet rays stably and uniformly, and a specific sterilizing apparatus is required. The applicant of the present invention pays attention to such a point, and repeats the operation of rotating and / or raising / lowering the immobilized enzyme in the washing / sterilization reaction tank, and irradiates the immobilized enzyme with ultraviolet light from thereover (Japanese Patent Application No. Hei. -43893).
【0005】[0005]
【発明が解決しようとする課題】本発明者らは、固定化
酵素の活性を低下させることなく、紫外線照射による殺
菌効果を高める方法について鋭意研究を重ねてきた。そ
して、長期間使用した固定化酵素に紫外線照射してその
殺菌効果を測定したところ、本来104 〜105 あるは
ずの殺菌力が実際には101 程度しかないことを見出し
た。そこで、この原因について検討し、(a)固定化酵
素に付着する微生物が重なり合っていること、(b)固
定化酵素が基質やその加水分解物などで層状あるいは膜
状に覆われていること、などの原因によって紫外線が微
生物に到達しないものと想定した。DISCLOSURE OF THE INVENTION The present inventors have intensively studied a method for increasing the bactericidal effect by ultraviolet irradiation without reducing the activity of the immobilized enzyme. Then, when the immobilized enzyme used for a long period of time was irradiated with ultraviolet rays and its bactericidal effect was measured, it was found that the bactericidal power which should originally be 10 4 to 10 5 is actually only about 10 1 . Therefore, the cause is examined, (a) that the microorganisms adhering to the immobilized enzyme overlap, (b) that the immobilized enzyme is covered with a substrate or a hydrolyzate thereof in a layer or film, It was assumed that ultraviolet rays did not reach the microorganisms due to such factors.
【0006】次いで、これらの障害を取り除くために超
音波処理を併用することを検討した結果、(1)紫外線
照射処理単独では101 程度の殺菌力であり、紫外線照
射量を増しても殺菌力は増加しないこと、(2)超音波
処理単独では30分間処理後に103 程度の殺菌力であ
り、処理時間を延長しても殺菌力は増加せず、洗浄効果
だけが得られること、(3)超音波処理を行った後、紫
外線照射処理を行うと殺菌力が急激に増大し、紫外線照
射量を4000mWs/cm2 とすると106 程度の殺
菌力であり、超音波処理と紫外線照射処理との相加効果
以上の効果が得られること、(4)超音波処理と紫外線
照射処理を併用した場合、超音波処理の時間が長いほう
が殺菌効果が増大すること等の知見を得た。Next, as a result of studying the use of ultrasonic treatment in combination to remove these obstacles, (1) the ultraviolet irradiation treatment alone has a sterilizing power of about 10 1 , and even if the ultraviolet irradiation amount is increased, the sterilizing power is increased. (2) The ultrasonic treatment alone has a bactericidal power of about 10 3 after the treatment for 30 minutes, the bactericidal power does not increase even if the treatment time is extended, and only the cleaning effect is obtained. ) After performing the ultrasonic treatment, the bactericidal power is sharply increased by performing the ultraviolet irradiation treatment, and the bactericidal power is about 10 6 when the ultraviolet irradiation amount is 4000 mWs / cm 2. (4) When the ultrasonic treatment and the ultraviolet irradiation treatment are used in combination, the longer the ultrasonic treatment, the longer the sterilization effect is obtained.
【0007】これらの知見から、紫外線照射処理単独で
殺菌力に限界があるのは、(a)微生物が重なり合い、
(b)基質などによって被覆されることに原因があり、
超音波処理と紫外線照射処理を併用するとそれらの妨害
要因が取り除かれ、紫外線が固定化酵素に到達して殺菌
効果を高めることができることを見出し、本発明を完成
するに至った。また、超音波処理を行う際に、界面活性
剤を含む液体中で処理を行うことにより、さらに殺菌効
果を高めることができることも見出した。したがって、
本発明は酵素活性を低下させずに固定化酵素に付着して
いる微生物を殺菌する方法を提供することを課題とす
る。[0007] From these findings, there is a limit to the bactericidal activity of the ultraviolet irradiation treatment alone because (a) microorganisms overlap,
(B) due to being coated with a substrate or the like,
When the ultrasonic treatment and the ultraviolet irradiation treatment are used in combination, it has been found that the obstructive factors are eliminated, the ultraviolet light reaches the immobilized enzyme, and the bactericidal effect can be enhanced, and the present invention has been completed. It has also been found that, when performing the ultrasonic treatment, the disinfection effect can be further enhanced by performing the treatment in a liquid containing a surfactant. Therefore,
An object of the present invention is to provide a method for disinfecting microorganisms attached to an immobilized enzyme without reducing the enzyme activity.
【0008】[0008]
【課題を解決するための手段】本発明の特徴は、固定化
酵素を殺菌する際に、超音波処理と紫外線照射処理を併
用することにある。さらには、超音波処理を行うに際し
て界面活性剤を含む液体中で処理を行うことにある。こ
のような処理を行うことにより、酵素活性を低下させず
に固定化酵素に付着している微生物を殺菌することがで
きる。A feature of the present invention resides in that, when sterilizing an immobilized enzyme, an ultrasonic treatment and an ultraviolet irradiation treatment are used in combination. Another object of the present invention is to perform the treatment in a liquid containing a surfactant when performing the ultrasonic treatment. By performing such treatment, microorganisms adhering to the immobilized enzyme can be sterilized without reducing the enzyme activity.
【0009】本発明の殺菌方法は、いかなる固定化酵素
にも適応できる。これらの固定化酵素としては、ラクタ
ーゼ、グルコースイソメラーゼ、プロテアーゼなどの酵
素をキトサン、キチン、樹脂など水不溶性の担体に化学
結合させるか、あるいはセルローストリアセテート、ポ
リアクリルアミドゲルなどに包括させて固定化したもの
を例示することができる。これらの固定化酵素は、カラ
ムなどに充填された状態であっても、撹拌槽に拡散され
た状態であっても、あるいはそれらの装置から取り出し
た状態であっても、超音波処理と紫外線照射処理を併用
することができる。[0009] The sterilization method of the present invention can be applied to any immobilized enzyme. As these immobilized enzymes, enzymes such as lactase, glucose isomerase, and protease are chemically bonded to a water-insoluble carrier such as chitosan, chitin, resin, or encapsulated in cellulose triacetate, polyacrylamide gel, or the like. Can be exemplified. These immobilized enzymes can be treated with ultrasonic waves and irradiated with ultraviolet rays regardless of whether they are packed in a column or the like, diffused into a stirred tank, or removed from those devices. The treatment can be used in combination.
【0010】超音波処理は、固定化酵素が充填あるいは
拡散している装置中に設置した超音波振動子か、装置か
ら固定化酵素を取り出して超音波洗浄器で、数分間〜6
0分間程度行う。この際、超音波処理を行う液体中に界
面活性剤を加えておくと、さらに洗浄効果を増大させる
ことができる。また、超音波処理を行う前に界面活性剤
を加えた液体で処理してもよい。本発明における界面活
性剤を含む溶液中で超音波処理とはこのいずれかの場合
であってもよい。ここで用いる界面活性剤は特に限定は
されず、グリセリン脂肪酸エステル、ソルビタン脂肪酸
エステル、ショ糖脂肪酸エステルあるいはレシチン等が
用いられる。グリセリン脂肪酸エステルはモノグリセリ
ン脂肪酸エステルであってもポリグリセリン脂肪酸エス
テルであってもよい。例えば、グリセリン脂肪酸エステ
ル、グリセリン酢酸脂肪酸エステル、グリセリン乳酸脂
肪酸エステル、グリセリンクエン酸脂肪酸エステル、グ
リセリンコハク酸脂肪酸エステル、グリセリンジアセチ
ル酒石酸エステル、グリセリン酢酸エステル等が、ソル
ビタン脂肪酸エステルとしてはソルビタンモノオレイン
酸エステル(スパン80)、ソルビタンモノラウリル酸
エステル(スパン20)等が、またショ糖脂肪酸エステ
ルとしては脂肪酸としてパルミチン酸、ステアリン酸を
含むエステルが用いられる。これらの界面活性剤は、通
常、0.5〜2.0重量%蒸留水に溶解して用いられ
る。これらの界面活性剤のなかでポリグリセリン脂肪酸
エステルは食品添加物として広く用いられている乳化剤
であり、安全性が高く、しかも洗浄効果を高めると共に
固定化酵素の活性を低下させないので本発明で使用する
のに特に好適である。[0010] The ultrasonic treatment is performed by using an ultrasonic vibrator placed in an apparatus in which the immobilized enzyme is filled or diffused, or by removing the immobilized enzyme from the apparatus and using an ultrasonic cleaner for several minutes to 6 hours.
Perform for about 0 minutes. At this time, if a surfactant is added to the liquid to be subjected to the ultrasonic treatment, the cleaning effect can be further increased. Before the ultrasonic treatment, the liquid may be treated with a liquid containing a surfactant. The ultrasonic treatment in the solution containing a surfactant in the present invention may be any of these cases. The surfactant used here is not particularly limited, and glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, lecithin and the like are used. The glycerin fatty acid ester may be a monoglycerin fatty acid ester or a polyglycerin fatty acid ester. For example, glycerin fatty acid ester, glycerin acetic acid fatty acid ester, glycerin lactic acid fatty acid ester, glycerin citrate fatty acid ester, glycerin succinic acid fatty acid ester, glycerin diacetyl tartaric acid ester, glycerin acetate, and the like. Span 80), sorbitan monolaurate (Span 20) and the like. As sucrose fatty acid esters, esters containing palmitic acid and stearic acid as fatty acids are used. These surfactants are usually used by dissolving in 0.5 to 2.0% by weight of distilled water. Among these surfactants, polyglycerin fatty acid ester is an emulsifier widely used as a food additive, and is used in the present invention because it has high safety, enhances the washing effect and does not decrease the activity of the immobilized enzyme. It is particularly suitable for
【0011】紫外線照射処理は、固定化酵素に付着する
微生物を殺菌できる程度の照度及び時間で行うが、通常
は照度10mW/cm2 前後で100秒間〜2000秒
間行い、照射量が1000〜20000mWs/cm2
となるように行うことが好ましい。特に照射量が300
0mWs/cm2 以上で効果を奏する。The ultraviolet irradiation treatment is carried out at an illuminance and time sufficient to kill microorganisms adhering to the immobilized enzyme. Usually, the irradiation is carried out at an illuminance of about 10 mW / cm 2 for 100 seconds to 2,000 seconds, and the irradiation amount is 1,000 to 20,000 mWs / cm 2. cm 2
It is preferable to perform so that Especially the irradiation dose is 300
The effect is obtained at 0 mWs / cm 2 or more.
【0012】超音波処理と紫外線照射処理の組み合わせ
は、超音波処理を行った後に紫外線照射処理を行っても
よく、また、超音波処理と紫外線照射処理を同時に平行
して行ってもよい。この際、従来知られている紫外線照
射方法あるいは処理装置、例えば、特開平1−2284
54や特願平2−43893に開示のものと超音波処理
を併用することができる。As for the combination of the ultrasonic treatment and the ultraviolet irradiation treatment, the ultraviolet treatment may be performed after the ultrasonic treatment, or the ultrasonic treatment and the ultraviolet irradiation treatment may be performed simultaneously in parallel. At this time, a conventionally known ultraviolet irradiation method or processing apparatus, for example, JP-A 1-2284
54 and Japanese Patent Application No. 2-43893 can be used in combination with ultrasonic treatment.
【0013】また、超音波処理や紫外線照射処理の前後
に、固定化酵素を蒸留水や緩衝液などで洗浄することに
よって、固定化酵素に付着している基質やその分解物、
あるいは微生物を除去しておくことが望ましい。Further, by washing the immobilized enzyme with distilled water or a buffer before and after the ultrasonic treatment or the ultraviolet irradiation treatment, the substrate adhering to the immobilized enzyme, its decomposed product,
Alternatively, it is desirable to remove microorganisms.
【0014】本発明の殺菌方法は、具体的には、牛乳中
の乳糖をグルコースとガクトースに分解する際に用いる
ラクターゼを担体に固定化した固定化酵素の殺菌、ある
いは乳糖、ホエー、脱脂乳などからオリゴ糖を生成させ
る際に用いるβ−ガラクトシダーゼを担体に固定化した
固定化酵素の殺菌などに用いることができる。さらに
は、蛋白質をペプチドや遊離アミノ酸に分解する際に用
いるプロテアーゼを担体に固定化した固定化酵素の殺菌
などにも用いることができる。[0014] The sterilization method of the present invention specifically includes sterilization of an immobilized enzyme obtained by immobilizing lactase used in decomposing lactose in milk into glucose and gactose on a carrier, or lactose, whey, skim milk and the like. Can be used for sterilization of an immobilized enzyme obtained by immobilizing β-galactosidase used for producing oligosaccharides from a carrier on a carrier. Furthermore, it can be used for sterilization of an immobilized enzyme in which a protease used for decomposing a protein into peptides or free amino acids is immobilized on a carrier.
【0015】[0015]
【発明の効果】本発明の効果について、試験例をあげて
説明する。The effects of the present invention will be described with reference to test examples.
【試験例1】酵素としてプロテアーゼS(天野製薬
(株)製)を用い、グルタルアルデヒドで架橋して担体
であるキトサンビーズ(商標名:キトパール、富士紡績
(株)製)に固定化し、固定化酵素(固定化プロテアー
ゼS)を調製した。この固定化プロテアーゼSをカゼイ
ンの加水分解処理に反復使用し、その結果、付着した微
生物の菌数が2.7×106 CFU/mlまでに増殖し
て汚染されたものを洗浄殺菌試験に供した。Test Example 1 Protease S (manufactured by Amano Pharmaceutical Co., Ltd.) was used as an enzyme, cross-linked with glutaraldehyde, immobilized on chitosan beads (trade name: Chitopearl, manufactured by Fuji Boseki Co., Ltd.), and immobilized. An enzyme (immobilized protease S) was prepared. This immobilized protease S was repeatedly used for the casein hydrolysis treatment. As a result, the contaminated microorganisms were grown to 2.7 × 10 6 CFU / ml and subjected to a washing and sterilization test. did.
【0016】上述のように汚染された固定化プロテアー
ゼSを出力100W程度の通常用いる超音波洗浄器で3
0〜60分間超音波処理し、それと同時平行して上方か
ら紫外線を照射した。この結果を表1に示す。残存微生
物の菌数は、対照に比べ、超音波処理と紫外線照射処理
を併用することによって顕著に低下し、特に、紫外線照
射量3000mWs/cm2 以上では殆ど微生物が残存
しなくなった。この結果から、固定化酵素の殺菌に超音
波処理と紫外線照射処理を併用すると、酵素活性を低下
させることなく、付着した微生物を殺菌することができ
ることが判明した。また、この殺菌効果は、紫外線照射
量3000mWs/cm2 以上で顕著であった。The immobilized protease S contaminated as described above is treated with an ordinary ultrasonic cleaner having an output of about 100 W for 3 hours.
Ultrasonic treatment was performed for 0 to 60 minutes, and at the same time, ultraviolet irradiation was performed from above. Table 1 shows the results. The number of remaining microorganisms was significantly reduced by using both the ultrasonic treatment and the ultraviolet irradiation treatment as compared with the control. In particular, when the ultraviolet irradiation amount was 3000 mWs / cm 2 or more, almost no microorganisms remained. From these results, it was found that when ultrasonic treatment and ultraviolet irradiation treatment were used in combination for sterilization of the immobilized enzyme, the attached microorganisms could be sterilized without reducing the enzyme activity. This bactericidal effect was remarkable when the irradiation amount of ultraviolet rays was 3000 mWs / cm 2 or more.
【0017】[0017]
【表1】 [Table 1]
【0018】[0018]
【試験例2】試験例1と同様の処理で汚染された固定化
プロテアーゼSを用い、界面活性剤の効果を確認するた
め、以下のような試料を調製し、固定化プロテアーゼS
の残存活性を測定した。Test Example 2 Using immobilized protease S contaminated by the same treatment as in test example 1, the following samples were prepared in order to confirm the effect of the surfactant.
Was measured for residual activity.
【0019】(1)試料1 使用済固定化プロテアーゼSを蒸留水中で出力100W
程度の通常用いる超音波洗浄器で超音波処理したもの。 (2)試料2 使用済固定化プロテアーゼSを界面活性剤としてグリセ
リン脂肪酸エステル・SYグリスターML750(商標
名、阪本薬品工業(株)製)を1重量%添加した蒸留水
中で出力100W程度の通常用いる超音波洗浄器で超音
波処理したもの。 (3)試料3 使用済固定化プロテアーゼSを界面活性剤としてグリセ
リン脂肪酸エステル・SYグリスターML750(商標
名、阪本薬品工業(株)製)を1重量%添加した蒸留水
中で出力100W程度の通常用いる超音波洗浄器で超音
波処理し、それと同時平行して上方から紫外線を照射し
たもの。(1) Sample 1 Spent immobilized protease S was output in distilled water at a power of 100 W
Ultrasonic treatment with a commonly used ultrasonic cleaner. (2) Sample 2 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner. (3) Sample 3 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above.
【0020】紫外線照度は平均5mW/cm2 であっ
た。結果を図1に示す。この図が示すように、界面活性
剤は酵素活性をほとんど低下させないことが分る。な
お、超音波処理60分間で固定化プロテアーゼSの残存
活性が初発より高くなった理由は、固定化プロテアーゼ
S表面に付着した加水分解物や微生物が除去されて、基
質との接触が円滑になったためである。The UV illuminance was 5 mW / cm 2 on average. The results are shown in FIG. As shown in this figure, it can be seen that the surfactant hardly reduces the enzyme activity. The reason that the residual activity of the immobilized protease S became higher than the initial activity in the ultrasonic treatment for 60 minutes was that the hydrolyzate and microorganisms attached to the surface of the immobilized protease S were removed, and the contact with the substrate became smooth. It is because.
【0021】[0021]
【試験例3】試験例1と同様の処理で汚染された固定化
プロテアーゼSを用い、界面活性剤の効果を確認するた
め、以下のような試料を調製し、固定化プロテアーゼS
表面に残存している微生物の菌数を計測することによっ
て、洗浄殺菌試験を行った。Test Example 3 Using immobilized protease S contaminated by the same treatment as in test example 1, in order to confirm the effect of the surfactant, the following sample was prepared and immobilized protease S
A cleaning sterilization test was performed by counting the number of microorganisms remaining on the surface.
【0022】(1)試料1 使用済固定化プロテアーゼSを蒸留水中で出力100W
程度の通常用いる超音波洗浄器で超音波処理し、それと
同時平行して上方から紫外線を照射したもの。なお、紫
外線照度は5mW/cm2 程度となるように調整した。 (2)試料2 使用済固定化プロテアーゼSを界面活性剤としてグリセ
リン脂肪酸エステル・SYグリスターML750(商標
名、阪本薬品工業(株)製)を0.5重量%添加した蒸
留水中で出力100W程度の通常用いる超音波洗浄器で
超音波処理し、それと同時平行して上方から紫外線を照
射したもの。なお、紫外線照度は5mW/cm2 程度と
なるように調整した。 (3)試料3 使用済固定化プロテアーゼSを界面活性剤としてグリセ
リン脂肪酸エステル・SYグリスターML750(商標
名、阪本薬品工業(株)製)を1重量%添加した蒸留水
中で出力100W程度の通常用いる超音波洗浄器で超音
波処理し、それと同時平行して上方から紫外線を照射し
たもの。なお、紫外線照度は5mW/cm2 程度となる
ように調整した。いずれの場合も処理時間は0〜60分
であった。(1) Sample 1 Spent immobilized protease S was output in distilled water at a power of 100 W
Ultrasonic treatment with a commonly used ultrasonic cleaner and irradiation of ultraviolet light from above in parallel with the ultrasonic treatment. The UV illuminance was adjusted to be about 5 mW / cm 2 . (2) Sample 2 About 100 W in distilled water to which 0.5% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with a commonly used ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above. The UV illuminance was adjusted to be about 5 mW / cm 2 . (3) Sample 3 Normally, about 100 W of output was used in distilled water to which 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo) was added using used immobilized protease S as a surfactant. Ultrasonic treatment with an ultrasonic cleaner, and simultaneous irradiation with ultraviolet light from above. The UV illuminance was adjusted to be about 5 mW / cm 2 . In each case, the processing time was 0 to 60 minutes.
【0023】その結果を図2に示す。この表にみられる
ように界面活性剤は殺菌処理時間の経過とともにその使
用量に応じて殺菌効果を表わし、特に50〜60分の処
理では生残菌数をほとんど0とする効果を奏する。FIG. 2 shows the results. As can be seen from the table, the surfactant exhibits a bactericidal effect according to the amount of the bactericidal treatment with the passage of the germicidal treatment time.
【0024】次に本発明の実施例を挙げて本発明を具体
的に説明する。Next, the present invention will be specifically described with reference to examples of the present invention.
【実施例1】直径6cm、長さ18cmのカラムに30
0mlの固定化プロテアーゼSを充填した後、ダウンフ
ローで10%カゼインを反応温度40℃、SV=3で連
続通液を行って加水分解物を調製した。時間の経過と共
に菌の生育が始まり、固定化プロテアーゼSへの乳タン
パク付着と細菌汚染が生じた。12時間通液後の残存酵
素比活性は70%であり、微生物菌数は106 オーダー
であった。このとき、カラムから固定化プロテアーゼS
を取り出し、pH7.0のリン酸緩衝液で洗浄して固形
分を除去した後、この固定化プロテアーゼSを殺菌槽に
移し、殺菌槽を回転させながら5mW/cm2 の紫外線
を60分間照射すると同時に出力100Wの超音波振動
子により超音波を照射した。このようにして処理した固
定化プロテアーゼSを無菌処理したpH7.0のリン酸
緩衝液で洗浄し、残存の酵素比活性は100%に回復
し、付着微生物の菌数0.0×101 であった。この固
定化プロテアーゼSを再びカラムに充填して、カゼイン
の加水分解に用いた。Example 1 A column having a diameter of 6 cm and a length of 18 cm was filled with 30
After filling with 0 ml of the immobilized protease S, a hydrolyzate was prepared by continuously passing 10% casein in a down flow at a reaction temperature of 40 ° C. and SV = 3. With the passage of time, the growth of the bacterium began, and milk protein adhered to the immobilized protease S and bacterial contamination occurred. The residual enzyme specific activity after passing the solution for 12 hours was 70%, and the number of microorganisms was of the order of 10 6 . At this time, the immobilized protease S
After removing the solid content by washing with a phosphate buffer of pH 7.0, the immobilized protease S was transferred to a sterilization tank, and irradiated with 5 mW / cm 2 ultraviolet rays for 60 minutes while rotating the sterilization tank. Simultaneously, an ultrasonic wave was emitted from an ultrasonic oscillator having an output of 100 W. The immobilized protease S thus treated was washed with a sterilized phosphate buffer of pH 7.0, the residual enzyme specific activity was restored to 100%, and the number of adherent microorganisms was 0.0 × 10 1 . there were. This immobilized protease S was packed again in the column and used for the hydrolysis of casein.
【0025】[0025]
【実施例2】直径6cm、長さ18cmのカラムに30
0mlの固定化プロテアーゼSを充填した後、ダウンフ
ローで10%カゼインを反応温度40℃、SV=3で連
続通液を行って加水分解物を調製した。時間の経過と共
に菌の生育が始まり、固定化プロテアーゼSへの乳タン
パク付着と細菌汚染が生じた。12時間通液後の残存酵
素比活性は70%であり、微生物菌数は106 オーダー
であった。このとき、カラムから固定化プロテアーゼS
を取り出し、pH7.0のリン酸緩衝液で洗浄して固形
分を除去した後、これを界面活性剤としてグリセリン脂
肪酸エステル・SYグリスターML750(商標名、阪
本薬品工業(株)製)を1重量%添加した蒸留水中で出
力100Wの超音波発振器を使用して超音波を照射する
と共に、殺菌槽を回転させながら5mW/cm2 の紫外
線を60分間照射した。このようにして処理した固定化
プロテアーゼSを無菌処理したpH7.0のリン酸緩衝
液で洗浄し、残存の酵素比活性は100%であり、付着
微生物の菌数0.0×101 以下であった。この固定化
プロテアーゼSを再びカラムに充填して、カゼインの加
水分解に用いた。Example 2 30 columns in a column of 6 cm in diameter and 18 cm in length
After filling with 0 ml of the immobilized protease S, a hydrolyzate was prepared by continuously passing 10% casein in a down flow at a reaction temperature of 40 ° C. and SV = 3. With the passage of time, the growth of the bacterium began, and milk protein adhered to the immobilized protease S and bacterial contamination occurred. The residual enzyme specific activity after passing the solution for 12 hours was 70%, and the number of microorganisms was of the order of 10 6 . At this time, the immobilized protease S
Was taken out and washed with a phosphate buffer of pH 7.0 to remove solids, and then 1% by weight of glycerin fatty acid ester / SY Glister ML750 (trade name, manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) as a surfactant. Ultrasonic waves were radiated using a 100 W output ultrasonic oscillator in distilled water to which% was added, and 5 mW / cm 2 ultraviolet rays were irradiated for 60 minutes while rotating the sterilizing tank. The immobilized protease S thus treated is washed with a sterile-treated phosphate buffer of pH 7.0, the remaining enzyme specific activity is 100%, and the number of adherent microorganisms is 0.0 × 10 1 or less. there were. This immobilized protease S was packed again in the column and used for the hydrolysis of casein.
【図1】試験例2の洗浄殺菌処理が酵素活性に及ぼす影
響を示す。FIG. 1 shows the effect of the cleaning and sterilizing treatment of Test Example 2 on enzyme activity.
─○─ 超音波処理 ─△─ 超音波処理と界面活性剤との併用 ─□─ 超音波処理、界面活性剤及び紫外線照射処理の
併用─ ○ ─ Ultrasonic treatment 併 用 Combination of ultrasonic treatment and surfactant ─ □ ─ Combination of ultrasonic treatment, surfactant and UV irradiation treatment
【図2】試験例3の界面活性剤の洗浄・殺菌補助処理効
果を示す。FIG. 2 shows the cleaning / sterilization assisting effect of the surfactant of Test Example 3.
─□─ 界面活性剤無添加 ─△─ 界面活性剤0.5%添加 ─○─ 界面活性剤1%添加 ─ □ ─ No surfactant added ─ △ ─ 0.5% surfactant added ─ ○ ─ 1% surfactant added
フロントページの続き (56)参考文献 特開 平1−228454(JP,A) 特開 昭63−237787(JP,A) 特開 昭61−12280(JP,A) 特開 平3−247269(JP,A) 実開 昭63−145500(JP,U) (58)調査した分野(Int.Cl.6,DB名) C12N 11/00 - 11/18Continuation of the front page (56) References JP-A-1-228454 (JP, A) JP-A-63-237787 (JP, A) JP-A-61-12280 (JP, A) JP-A-3-247269 (JP) , A) Actually open 63-145500 (JP, U) (58) Fields investigated (Int. Cl. 6 , DB name) C12N 11/00-11/18
Claims (4)
理とを併用して固定化酵素に付着する微生物を殺菌する
ことを特徴とする固定化酵素の殺菌方法。1. A method for disinfecting an immobilized enzyme, comprising using an ultrasonic treatment and an ultraviolet irradiation treatment together with the immobilized enzyme to sterilize microorganisms adhering to the immobilized enzyme.
行う請求項1に記載の固定化酵素の殺菌方法。2. The method for sterilizing an immobilized enzyme according to claim 1, wherein the ultrasonic treatment is performed in a liquid containing a surfactant.
である請求項2に記載の固定化酵素の殺菌方法。3. The method according to claim 2, wherein the surfactant is a glycerin fatty acid ester.
2 以上の紫外線照射量で行う請求項1乃至3のいずれか
に記載の固定化酵素の殺菌方法。4. An ultraviolet irradiation treatment is performed at 3000 mWs / cm.
The method for sterilizing an immobilized enzyme according to any one of claims 1 to 3, wherein the method is performed with two or more ultraviolet irradiation doses.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP3075839A JP2832760B2 (en) | 1990-09-07 | 1991-03-15 | Sterilization method of immobilized enzyme |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP23823590 | 1990-09-07 | ||
JP2-238235 | 1990-09-07 | ||
JP3075839A JP2832760B2 (en) | 1990-09-07 | 1991-03-15 | Sterilization method of immobilized enzyme |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH04218371A JPH04218371A (en) | 1992-08-07 |
JP2832760B2 true JP2832760B2 (en) | 1998-12-09 |
Family
ID=26416997
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP3075839A Expired - Lifetime JP2832760B2 (en) | 1990-09-07 | 1991-03-15 | Sterilization method of immobilized enzyme |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP2832760B2 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6572820B2 (en) | 1996-02-05 | 2003-06-03 | Asahi Medical Co., Ltd. | Sterilization-protecting agent and sterilization method |
US5997907A (en) * | 1997-03-12 | 1999-12-07 | Rhodia Inc. | Enhancement of guar solution stability |
ATA37099A (en) * | 1999-03-04 | 2000-02-15 | Lactoprot Alpenlaendische Milc | METHOD FOR ENZYMATIC LACTOSE CLEAVING, ESPECIALLY BY MEANS OF MEMBRANE DIFFUSION REACTORS |
EP1852443A1 (en) | 2006-05-05 | 2007-11-07 | Leukocare AG | Biocompatible three dimensional matrix for the immobilization of biological substances |
CN107252108B (en) * | 2017-06-27 | 2021-01-29 | 福建农林大学 | Lactase microcapsule and preparation method thereof |
CN112280638A (en) * | 2019-07-24 | 2021-01-29 | 宁波晨实生物科技有限公司 | Method for preparing raw rice wine by utilizing ultrasonic sterilization |
-
1991
- 1991-03-15 JP JP3075839A patent/JP2832760B2/en not_active Expired - Lifetime
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Publication number | Publication date |
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JPH04218371A (en) | 1992-08-07 |
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